The effects of partial sleep deprivation and the Sub-maximal NDKS exercise testing protocol on

Copyright: © Saghiv M (2018). This Article is distributed under the terms of Creative Commons Attribution 4.0 International License *Corresponding Author(s): Moran S Saghiv Associate Professor, Exercise Physiology Department, Casey Center, Room 141B, University of Mary, 7500 University Drive, Bismarck, ND 58504, USA Tel: 702-908-2390, Fax: 701-255-7687 Email: mssaghiv@umary.edu Annals of Cardiology and Vascular Medicine


Introduction
It has long been known that different extents of sleep deprivation (partial and/or full) have negative implications on function, hemodynamic, and physiological responses [1][2][3][4][5][6]. The influences of sleep deprivation may in some cases, be sex-specific (male vs female). Such influences may be characterized by neurologic, endocrine, physiologic-metabolic, and behavioral changes, often for worse [7][8][9].
Prior studies pertaining to the influences of sleep deprivation of different extent clearly indicate that while sleep deprived, multiple aspects of function, across several systems are influenced for the worse. Such influences may include endothelial dysfunction, hypertension, decreased brain activity, cardiac dysfunction, heart rate variability, arrhythmias, blood pressure, renal function, and more [10][11][12][13].
On the other hand, exercise and adaptation to exercise of various exercise modalities such as aerobic training, and resis-Annals of Cardiology and Vascular Medicine tance training has been proven to negate part and/or all of the adverse effects of sleep deprivation in humans and rats [14][15][16][17].
Klotho is an enzyme that in humans is encoded by the KL gene [18]. S-klotho is a type I membrane protein first documented in 1997 [19]. The protein is associated with the degenerative process, acceleration of aging, bone lose, and alcohol consumption [20][21][22][23][24]. Furthermore, s-klotho is a role-player in determining the sensitivity to Insulin, mediates the binding of FGF19, FGF20, and FGR23 to their receptors as part of the growth process at the cellular level [23].
In addition, S-klotho is suggested as a cardiovascular system "protector" via means of endothelium-derived NO production [25][26]. S-klotho has been proven to affect cellular Calcium homeostasis via increased expression of TRPV5, and decreased TRPC6; and increases membrane expression of Inward Rectifier ROMK; in mice, under-expression causes hypervitaminosis of Vitamin D; altered mineral-ion homeostasis resulting in accelerated aging; a syndrome of accelerated aging; arteriosclerosis; impaired endothelium-dependent vasodilation; and impaired angiogenesis [23,[27][28][29][30].
To the best of the authors' knowledge, no prior published data exists as to the influence of partial (less than 40 hours of lack of sleep) and/or full sleep deprivation (more than 40 hours of lack of sleep) on s-klotho in humans and/or during and after an exercise test of any sort. Thus, the aim of this study was to investigate the influences of partial sleep deprivation (20 hours of lack of sleep) on s-klotho concentration (pg·mL -¹), heart rate (bpm), blood pressure (mmHg), fasting blood glucose (mg·dL -1 ), Lactate (mmol·L -1 ), Oxygen saturation (O 2 sat, %) and rate of perceived exertion (RPE, Scale) before and after a Nustad, Dressler, Kobes, Sagiv (NDKS) sub-maximal exercise testing protocol in young, healthy, and trained men.

Overview, subjects, recruitment and enrollment:
After achieving Institutional Review Board (IRB) approval to conduct this study, students at the University of Mary, Bismarck, ND, USA, were asked to participate in the study. Twenty males, 21.6±1.82 years of age, volunteered to participate in this study. All participants were healthy and trained individuals.
Subjects visited with the researcher 3-4 times. Upon agreeing to participate, subjects received an informed consent form via email and/or hard-copy, read the form, and indicated in a clear manor they have fully understood its content, prior to signing the form. The subjects were given as many opportunities as needed and/or wanted to ask questions pertaining to the study's purpose, protocol, etc.
Subjects then filled-out a Health History Questionnaire (HHQ) and signed the HHQ. The information in the HHQ and followup questions (if raised) were used to determine rather or not a specific prospective subject were to be included or excluded from the study. Prospective subjects were excluded if they had diagnosed musculoskeletal injury, cardiovascular disease, pulmonary disease, mental health problems, endocrine instability, and/or were treated with medication for any reason.

Baseline measurements:
Following inclusion in the study (as part of the first visit with the researchers), the subjects 'heart rate, blood pressure, O 2 sat, fasting blood glucose, blood lactate, and a 5mL blood sample (from the Median Cubital Vein) were obtained, recorded on file and/or labeled. Prior to leaving the lab, subjects were randomly assigned to their two sub-maximal NDKS exercise tests. Using a table of random numbers, half of the subjects were assigned to be tested Non-partially Sleep Deprived first (NSD), and the other half, scheduled to be tested Partially Sleep Deprived first (PSD). Subject were allowed to leave the lab in the absence of adverse reactions and/or return to baseline values.

Prior to exercise testing sessions:
Subjects were instructed to completely avoid alcohol consumption of any sort, moderate/intensive exercise of any sort, caffeine, use of any over-the-counter medications, and/or stressful situations at least 8 hours prior to data collection. Subjects self-reported their compliance with these instructions.

Exercise testing (NSD or PSD):
Subjects tested while partially sleep deprived, reported to the lab in 30-minute increments starting at 2am (up to six subjects were tested per data collection session; two at a time, and no more than three pairs altogether). Subjects were instructed to be completely awake from 6am the prior day. Subjects were contacted via email, text message, and/or phone call the day before testing to assure compliance with the instructions.
Subjects tested while non-partially sleep deprived, reported to the lab in 30-minute increments starting at 6am (up to six subjects were tested per data collection session; two at a time, and no more than three pairs altogether). Subjects were instructed to sleep 7.5-8 hours prior to testing. Subjects contacted via email, text message, and/or phone call the day before testing to assure compliance with the instructions.
Upon arrival at the lab, the subjects were asked if they have complied with the instructions (one after the other). Subjects non-compliant with the instructions were rescheduled; all subjects were tested twice within a period of no more than two weeks in between test, and for the vast majority of cases, within a week a part.
Subjects complaint and without signs of a health condition (cold, influenza, etc.) were seated for 5 minutes, doing nothing. Afterwards, Heart rate, and blood pressure were obtained and compared to the subject's baseline values. The subject then continues to a warm up, followed by a NDKS sub-maximal exercise test (see appendix A for actual protocol), and dynamic recovery following the test itself. The exercise test was terminated once the subjects achieved 85% of his age-estimated maximal heart rate according to the equation [HR 85% = 0.85×(220-age)] or if the subject wished so for any reason. Heart rate, blood pressure, RPE (6-20 scale), and O 2 sat were obtained every other minute during the exercise test (see appendix A). S-klotho was obtained after subjects returned to resting heart rate and blood pressure values post-dynamic recovery that included waking at 1.7 miles per hour at 0% grade. Annals of Cardiology and Vascular Medicine Samples and measurements: 5mL blood samples were obtain (according to universal precautions for blood borne pathogens) from the Median Cubital Vein and stored in BD Vacutainer™ Venous Blood Collection Tubes: SST™ Serum Separation Tubes with Conventional Stopper until analyzed; samples were then refrigerated.
Subject without adverse reactions and upon return to the vicinity of baseline values were allowed to leave the lab.
Blood samples were analyzed via Soluble Klotho (Human serum) ELISA Kit SK00708-08 (Adipo Bioscience, Inc.), with a Standard range of 313-20000 pg·mL -1 ; Sensitivity of 80 pg·mL -1 ; Intra-CV of 4-6%; and Inter-CV of 8-10%. Blood pressure was obtained utilizing an Omron sphygmomanometer; heart rate via FS1 Polar Heart Rate Monitor and strap, Oxygen saturation was obtained via Oxywatch C20 ChoiceMMed pulse-oximeter; Lactate and glucose (mg·dL -1 ) were obtained via Point-of-Care Testing using a Nova Biomedical Lactate Plus meter and a Health Mart True Metrix meter respectively. The treadmill used in this study was a SportsArt Fitness 6320.

Statistical analysis:
SPSS 23 for Windows was used to analyze the data, Comparisons between conditions (partial sleep deprivation vs non-partial sleep deprivation) were conducted via One-way repeated measures ANOVA and the Tukey post hoc test. Significance was set at p < 0.05. Results are presented as means ± standard deviation, F values when appropriate.

In general:
All subjects participated in this study without any prolonged adverse reactions. Some subjects had a temporary reaction to their blood being drawn (dizziness, elevated heart rate, hypertension, hypotension, and in two cases subjects reacted with syncope). All subjects left the lab without any adverse clinical signs.

Baseline measures:
All comparisons, regardless of condition, between immediate-post and post-dynamic recovery concentrations and baseline concentration of s-klotho were significant (p≤0.05). All possible comparisons pertaining to measures of Glucose were statistically non-significant (p>0.05). Comparisons between conditions of peak heart rate, systolic blood pressure, diastolic blood pressure, rate of perceived exertion, and oxygen saturation, were all non-significant (p>0.05).

S-klotho:
S-klotho concentration at baseline was 430.31±12.62 (pg·mL -¹). In the absence of sleep deprivation (NSD), s-klotho concentrations increased to 557.97±20.08 immediate post (85% of age-estimated heart rate maximum). S-klotho concentrations then decreased to 453.29±4.34 post dynamic recovery.  NSD: Non-Sleep Deprived; PSD: Partially Sleep Deprived; SD: Standard Deviation; †: Significant difference compared to baseline under the same condition; ‡: Significant difference compared to immediate post under the same condition; α: Significant difference between conditions for the same timing of obtaining. Heart rate:
Immediate-post lactate while non-sleep deprived and while partially sleep deprived were found to be significantly higher in comparison to baseline [2.09±1.26 vs 5.63±2.6mmol·L -1 , respectively, (F(39,1)
Oxygen saturation increased in the transition from immediate-post to post-dynamic recovery in both NSD and PSD conditions (97.1±0.91 vs 96.37±1.34%, respectively). No significant difference was found between post-dynamic recovery oxygen saturation in comparison between NSD and PSD conditions

Discussion
This study is a first of a kind in regards to partial sleep deprivation and its influences on s-klotho levels in men before and after sub-maximal exercise testing in men. The researchers are unaware of published data pertain to the topic. With that said, s-klotho is a biomarker of aging and anti-aging alike, dependent on the extent of gene expression [32,33]. Thus, this study starts to shade light on the connection between partial sleep deprivation and aging/anti-aging as it is represented via s-klotho concentration under certain conditions. Sleep deprivation (acute and/or chronic) is well documented as an influencer of function and performance in both healthy and clinical populations, across sex and age to different extents [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17].
The present study demonstrates a clear and significant influence of partial sleep deprivation on s-klotho responses to the sub-maximal NDKS exercise test in young, healthy, and trained men, while compared to being non-sleep deprived. The data of this study suggest that partial sleep deprivation of 20 hours, is a strong enough stressor, resulting in lower levels of s-klotho immediate-post, and post-dynamic recovery, while compared to those of the non-sleep deprived state.
Partially sleep-deprived, and from baseline to 85% of ageestimated heart rate maximum, subject were unable to elevate s-klotho levels, although the same subject were able to do so while non-sleep deprived. Though the design of the study does not allow proving the reason for this dynamic, it is the researchers' suggestion that the inability to elevate s-klotho as much as while non-sleep deprived represents an impaired reaction, induced by fatigue.
Proper sleep has been indicated as an anti-oxidative factor [34,35]. On the other hand, exercise induces the creation of reactive oxygen species (ROSs) [36][37][38]. Thus, it is possible that the need to elevate s-klotho during exercise is partially in order to assist with the need to battle ROSs. The impaired responseis further apparent at post-dynamic recovery, whereas the level during non-sleep deprivation return almost completely to baseline, most likely indicating the ability to counteract ROSs better than while partially sleep deprived.
Baseline heart rate values for this group of participants were within the range of previously reported findings for age, training status, and sex matching subject.  reported resting heart rate to be 68.7±9.3 bpm [39] while Papathanasiou et al. (2013) reported resting heart rate to be 66.3±6.1 bpm [40]. Both resting systolic and diastolic blood pressure values of the subjects in this study were within normal and healthy ranges [41].
Subjects' average resting fasting blood glucose was within the glucose range considered to indicate being pre-diabetic (100-126 mg·dL -1 ) [42]. While matching age, sex, and training status subjects have been found to be pre-diabetic [43], it has been reported that roughly 1.7% of men ages 20-30 years are pre-diabetic [43]. Thus, while it is theoretically possible that all of the current study's subjects fell within that 1.7%, it is highly unlikely. It is for this reason suggested that the higher baseline glucose levels of the current study's subjects are due to possible non-compliance with the pre-data session collection instructions.
Baseline lactate concentration was within the previously reported range at rest for sex, age, and training status (0.5-2.2 mmol·L -1 ) [44].

Annals of Cardiology and Vascular Medicine
Oxygen saturation measured via pulse-oximeter showed that subjects' oxygenation levels were adequate and contradicted a diagnosis of hypoxemia [45]. These results are similar, yet slightly lower than those reported by [46] whereas oxygen saturation was 98.53±0.52%.
Subjects of this study have been tested in a sub-maximal treadmill exercise test, and have achieved greater heart rate peak values than previously reposted. Patrick et al. (2017) found no significant influence of sleep deprivation on peak heart rate in young, healthy, male students undergoing a sub-maximal cycling test (149±22 vs 146±20 bpm, p=0.079, respectively)in comparison to being non-sleep deprivedIn both studies, heart rate peak under partial sleep deprivation was insignificantly lower [1]. Patrick et al. (2017) additionally reported blood pressure peak post-exercise to be significantly different between conditions of sleep deprivation and non-sleep deprivation, whereas all other variable were not significantly different (135±12 vs 140±17 mmHg, p=0.012, respectively). While the results of Patrick et al. (2017) are statistically significant, the values achieved are significantly lower in comparison to this study's values for heart rate peak. Both differences in pick heart rate and systolic blood pressure between the current study and those of Patrick et al. (2017) may be explained by the extent of active muscle mass involved and the cardiac output demanded (treadmill vs cycle ergometer) [1].
The findings of this study as they pertain to RPE contradict prior findings whereas sleep deprivation resulted in elevated RPE. An interesting finding of these studies is the fact that physiological measures where not significantly affected while RPE increased, perhaps hinting that the cognitive and psychological component outweighs the physiological aspect [2]. While RPE was higher while partially sleep deprived, the differences were not great enough to induce a statistical significance. Similar insignificant finding regarding RPE were reported by Partick et al (2017) [1].
Recent data suggests that Oxygen saturation is not significantly altered due to partial sleep deprivation during exercise testing. Such findings were reported while investigating the effects of two types of Partial Sleep deprivation (PSD) on biomarkers of muscle and cardiac injuries in response to acute intermittent exercise in professional athletes [4]. These findings align perfectly with the findings of this study.
Prior studies have shown that partial sleep deprivation has no significant effect on Glucose, yet 48 hours of sleep deprivation (full sleep deprivation) results in a 40% greater Insulin resistance level [5]. While this study did not investigate the influences of partial sleep deprivation on Insulin and/or Insulin resistance, the findings of this study align with those of prior studies as to the insignificant effects of partial sleep deprivation on fasting blood glucose levels [5].
Very few data exist regarding the influences of sleep deprivation on Lactate and/or Lactic Acid. The findings of this study contradict a previous report whereas partial sleep deprivation caused a significant increase in Lactate [6].

Conclusions
The data of this study suggest that partial sleep deprivation is a strong enough stimulus affecting s-klotho concentration in response to the sub-maximal NDKS aerobic exercise testing pro-Annals of Cardiology and Vascular Medicine tocol. Our results further support the possibility of an impaired ability to elevate s-klotho as a result of sub-maximal exercise, and impaired ability to return to baseline levels post-dynamic recovery.
The results of this study fairly align with the majority of existing data pertaining to the lack of significant influences of partial sleep deprivation on physiological variables, all but RPE.